Fish growth hormones

ABSTRACT

The invention relates to new fish growth hormones, nucleic acids encoding them, and transgenic fish that express them.

RELATED APPLICATIONS

[0001] This is a continuation of Application Ser. No. 09/549,831, filedon Apr. 14, 2000.

BACKGROUND OF THE INVENTION

[0002] Farmed fish, such as ayu or grouper, are commercially valuablecrops. A means of increasing the growth rate or size of fish crops istherefore commercially useful.

SUMMARY OF THE INVENTION

[0003] The invention is based on the discovery of three new fish growthhormone genes (from Plecoglossus altivelis, Epinephelus awoara, andDanio rerio) that can be used to increase fish crop production. Forexample, each of the new fish growth hormone genes can be used togenerate transgenic fish expressing a fish growth hormone of theinvention. These fish will exhibit greater growth rates and body sizesthan the parent fish from which the transgenic fish was derived.Alternatively, the growth hormones can be recombinantly produced andintroduced into the fish diet (see, e.g., Ben-Atia et al., Gen. Compar.Endocrinol. 113:155-164, 1999).

[0004] In addition, the growth hormone polypeptides and fragmentsthereof can be used to generate antibodies that specifically bind to agrowth hormone of the invention. These antibodies can be used to, e.g.,detect fish pituitary tissue in a sample. Other fragments, such as thesignal sequence, can be fused to heterologous proteins to render themsecretable. The growth hormone cDNA and fragments thereof can be used toscreen DNA libraries (e.g., genomic or cDNA libraries) to isolate otherrelated genes and the growth hormones which they encode.

[0005] The sequences of the new fish growth hormones are given below:Plecoglossus altivelis (common name: ayu) GGCACGAGCA GAGACCAGCGATTCACCCAG AGTTCTCTAC TGACGACATC AGATACGACA (SEQ ID NO:2) AGGACAGAATGGGGCAAGTG CTTCTCCTGG TGACCCTCCT GCTGGTCAGT GACCTGGTCA          M  G  Q   V  L L   L  V  T  L   L L   V S   D L  V GATCTGCATC CGGCTCAGAGAACCAGCGTC TCTTCAGCAT CGCTGTCAAT CGTGCTCAAC R S   A  S   G  S   E N Q   R  L  F  S   I  A  V N   R A  Q ACCTGCACCT GCTGGCCCAG AAGATGTTCAACCACTTTGA GGGAAACCTC TCTCCAGATC H L   H  L   L  A   Q K M   F  N  D   F  E  G  N  L   S P  D ATCGAAGGCA GATGAATAAG ATCTTCCTGCAGGACTTCTG TAACTCAGAC TCCATCATCA D R   R  Q   M  N   K I  F  L  Q  D   F  C  N  S D   S I  I CCCCCGTGGA CAAGCACGAG ACTCAGAAGAGCTCGGTGCA GAAGTTGCTC CACATCTCGT S P   V  D   K  H   E T  Q  K  S  S  V   Q K   L L   H I S TCCGTCTGGT GGAGTCGTGG GAGTACCCCAGCCAAGCTCT CAGCAGCTCA CTGAGCCTCA F R   L  V   E  S   W E Y   P  S  Q  A   L  S  S  S   L S L GTCGCTTCAG TGAGATCCCT CTGAAACTCACCGACCTGAA GCTGGGCATC GACACCATCC S R   F  S   E  I   P L  K  L  T  D  L   K L   G  I   D T  I TCAGGGGTAC CCAAGATGGG CTCCTCAGCCTGGAGGATAA TGAGGCCCAG CAGCTGCCCC L R   G  T   Q  D   G L  L  S  L  E   D  N E   A  Q   Q L  P CCTATGAGAA TTACTACCAG AACCTCGGGGGTGACGACAA CATCCACAGG AGCTACGAGC P Y   E  N   Y  Y   Q N L   G  G  D  D   N I   H R   S Y E TGCTAGCCTG CTTCAAGAAA GACATGCACAAGGTGGAAAC GTACCTGACT GTTGCAAAGT L L   A  C   F  K   K D M   H  K  V   E  T Y   L  T   V A K GTCGTCGATC CCTGGAAGCC AACTGTACCCTGTAGAACCA CGCCTGACAG ACAGAACCAA C  R  R  S   L  E   A  N C   T  L   *TCTATCTCAG ATCAACTTAG CCCTGGACTG TGTCACATTT AATATCCTCC CCCCTCCATA (SEQID NO:1) TTCCATACTT TCTGCTTTGG AATGAAACTA TATAAATAAA CCAACGTGTATTTACAAAAA GTATAACTAT ATATTTTGTT AAGGTTCGTT TGAAAGCAGA AGGAATGGAATCTCTCAAGA CGACAGAGAG GGAAGGATGT CGCAATCCAA CGTTGTTTTG TTTTGAAGTGTAGGTTTGTT CTGAGACACC AGGCTCATGG TTTTCCATCT TACCAACAAC AGCACTTTCTATACGTCTGT CTCTTTTCCT ATTAGCAGAG TCTGATTAAG GATATTTCAG CCAGATAAGTGTTTCTAATG GAGGTGAATT AGTTTGTTAC TCGCAAACGA TTCAAGATAC AATTCATATTGGTGCTCATT TTCATTAGAA ATCAAAACAT ACTAACTCAC AGAAGATCTA TGTTTTAATAACAGTTTATG TCCATTTGGG CACATTGATA ATGATTGCCT GTCGATGAAA AGACTCCCATATGTATTTGT GTTTTGTTTT TTAAGTATTA AGAGATGTTT TGTCATGAAG ACATCTTTCTACTTGATTCA TTTGCCTCTC CTTTTTACTT TACGTCAAAT GTGTCATTGT TTTTAATTTCTAATAAAGCT GTTGTTTATT GCAAAAAAAA AAAAAAAAAA

[0006] The leader peptide sequence (SEQ ID NO:3) is shown in bold, andthe polyA signal is underlined. The nucleotide sequence between theinitiation codon and the polyA signal, excluding the initiation codonand the polyA signal, is designated SEQ ID NO:4. Epinephelus awoara(common name: yellow grouper) ACGAGCTCAG ACCTGATCCA CCAGAGCCAGACCTGATCCC CCAGAGCCAG ACCTAATCCC (SEQ ID NO:6) AGACCAGCCA TGGACCGAGTCGTCCTCCTG CTGTCAGTAG TGTCTCTGGG CGTTTCCTCT          M   D  R   V V L   L  L  S   V  V   S L  G  V  S S CAGCCAATCA CAGACGGCCA GCGACTGTTCTCCATCGCTG TCAGCAGAGT TCAACATCTC Q P   I  T  D   G   Q R L   F  S  I   A  V   S R V  Q  H L CACCTGCTTG CTCAGAGACT CTTCTCCGACTTTGAGAGCA GTCTGCAGAC AGAGGAGCAG H L   L  A  Q   R   L F  S  D  F  E  S   S   L Q T  E  E  Q CGACAGCTCA ACAAGATCTT CCTGCAGGACTTTTGTAACT CTGATTACAT CATCAGCCCC R Q   L  N   K  I   F L  Q  D  F  C   N  S   D Y I  I  S P ATTGACAAGC ATGAGACGCA GCGCAGCTCCGTGTTGAAGC TGTTGTCGAT CTCCTATCGG I D   K  H   E  T   Q  R S  S  V  L   K  L  L  S  I  S  Y R TTGGTGGAGT CCTGGGAGTT CCCCAGTCGGTCCCTGTCCG GAGGTTCTGC TCCCAGAAAC L V   E  S   W  E   F P  S  R  S  L  S   G   G S A  P  R N CAGATTTTTC CCAAACTGTC TGAATTGAAAACTGGGATCC TGCTGCTGAT CAGGGCCAAT Q I   F  P   K  L   S E L   K  T  G   I  L   L L  I  R  A N CAGGACGGAG CGGAGCTCTT TCCTGACAGCTCCGCCCTCC AGTTGGCTCC TTATGGGAAC Q D   G  A   E  L   FP  D   S  S  A  L    Q   LAP Y GN TATTATCAGA GTCTGGGCGC AGACGAGTCGCTGCGACGAA CGTACGAACT GCTGGCGTGT Y Y   Q  S   L  G   A D E   S  L  R  R   T   Y E L L A C TTCAAGAAAG ACATGCACAA GGTGGAGACCTACCTGACGG TGGCTAAATG TCGACTCTCT F K   K  D   M  H  K V  E  T  Y  L   T  V   A K  C  R  L S CCTGAGGCCA ACTGTACCCT GTAGCCCCGCCTCTCCAGTA TGAAGACAAG CCCCCATGTG P E  A   N   C  T L  * GATGATGTAATGCTGTGTGT TCTGTAGTCC CGCCCACATG TTTTCTGACT CTGCTAATTA (SEQ ID NO:5)GCATTAGTGT TAGCCACAGT GTTAGCCTGT CTTCAGTGGT TTGTTGGAGC AGGTGTTATTATGATGACAG CCGTCGACAG GAACTOATGT CATTTTGTCA CCATGTGTAA TAAAGTGTGTGCTGTGTTGC ATTCAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAAAAAAAAAAAA AA

[0007] The leader peptide sequence (SEQ ID NO:7) is shown in bold, andthe polyA signal is underlined. The nucleotide sequence between theinitiation codon and the polyA signal, excluding the initiation codonand the polyA signal, is designated SEQ ID NO:8. Danjo rerio (commonname: zebrafish) CCTTCAATCA AGAACGAGTT TGTCTATCTT GGACAAAATG GCTAGAGCATTGGTGCTGTT (SEQ ID NO:10)                                      M  A  A  A  L  V L   L GCAGTTGGTGGTGGTTAGTT TGCTGGTGAA TCAGGGGAAA GCCTCCGAAA ACCAGCGGCT  Q L   V V   V   S L L   V  N   Q   G K  A S   D  N  Q  R  L CTTCAACAACGCAGTCATCC GTGTGCAACA CCTTCACCAG CTGGCTGCAA AAATCATTAA  F N   N A   V   I R V   Q  H   L  H  Q  L  A  A  K  M I   N CGACTTTGAGGAAGGTCTTA TGCCTGAGCA ACGCAGACAG TTGAGTAAAA TCTTCCCTCT  D F   E  E  G  L  M P   E  E   R   R  Q  L  S  K  I  F P   LGTCGTTCTGC AACTCTGACT CCATCGAGAC GCCGACGGGA AAAGATGAAA CGCAAAAAAG  S F   C  N  S   D S I   E  T   P  T  G  K D   E  T  Q K   S CTCTATGTTGAAGCTGCTTC GTATCTCTTT CCGCCTCATT GAATCCTGGG AGTTTCCCAG  S M   L  K  L  L  R I  S   F   R  L  I  E S   W  E  F P   S CCAGACCTTGAGCTCCACTA TCTCAAACAG CCTGACCATC GGAAACCCCA ACCAAATCAC  Q T   L  S  S   T I S   N  S   L   T I  G N   P  N  Q L   T TGAGAAACTGGCGGACCTGA AAATGGGCAT CAGCGTGCTC ATCAAGGGAT GTCTCGATGG  E K   L  A  D   L K M   G  I   S  V  L  I K   G  C  L D   G ACAGCCAAATATGCATGACA ACGACTCCCT GCCCTTGCCT TTTGAGGATT TCTACCTGAC  Q P   N M   D   D N D   S  L   P  L  P  F  E  D  F  Y L   T CGTAGGGGAGACCAGTCTCA GAGAGAGCTT TCGCCTGCTG GCCTQCTTCA AGAAGGACAT  V G   D  T  D  L  R E   S  F   R  L  L  A  C  F  K  K D   M GCACAAGGTGGAAACTTACC TGAGGGTTGC GAATTGCAGG AGATCTCTGG ATTCCAACTG  H K   V  E  T   Y L R   V  A   N   C R  R  S  L  D  D N   C TACCCTGTAGAGGCCGCTAA TGTATTGCTA GTCAAAGCCT GCTTTATCCT TTTCTGCAAA   T L   *TCTAAGACCA GTTTGCATTA TCAAAACATA AACTAATTAT TATCTGGTCC TATATATGCA (SEQID NO:9) GGAAATATCA AGCAGGCATG GCTGGATCTG TACTTTATTT CCCTTCCATAAACCTTACAC CTACCACCAT TGTATTTATT CTTCTTATTG GGAAGTATTA TCATTTCAAGATGTTCCTTA AAAACGTAAA TATTGATTCT TATTTAATAT CCGAACCTTA TTCACAGTGGTGCTTAGCAA TTTCTGGCGA TATTTTCTTA AATGTGCCAA AATTGACTTA AATCAAAGTGCTAATATTGT GCTTTGGTGT ATATTATATC TAAAACAGTT AAAGATCAGT GTTCAAAGGGTTCACTCCCA AATGTGTGAA TGGAAACGTG TCTGTCTGAT AGATTCTTGC CTTPATATTATCAACTCATC CTGTTCTATT CTAACTGTAT CAATTAAAGT TTTAAAATGC AAAAAAAAAA AAAAA

[0008] The leader peptide sequence (SEQ ID NO:11) is shown in bold, andthe putative polyA signal is underlined. The nucleotide sequence betweenthe initiation codon and the polyA signal, excluding the initiationcodon and the polyA signal, is designated SEQ ID NO:12.

[0009] The mature growth hormone peptides of zebrafish, ayu and yellowgrouper are shown below: Zebrafish growth hormoneSDNQRLFNNAVIRVQHLHQLAAKMINDFEEGLMPEE (SEQ ID NO:19)RRQLSKIFPLSFCNSDSIETPTGKDETQKSSMLKLLRISFRLIESWEFPSQTLSSTISNSLTIGNPNQLTEKLADLKMGISVLIKGCLDGQPNMDDNDSLPLPFEDFYLTVGDTSLRESFRLLACFKKDDMHKVETYLRVANCR RSLDSNCTL Avu growth hormoneSENQRLFSIAVNRAQHLHLLAQKMFNDFEGNLSPDD (SEQ ID NO:20)RRQMNKIFLQDFCNSDDIIDPVDKHETQKSSVQKLLHISFRLVESWEYPSQALSSSLSLSRFSEIPLKLTDLKLGIDTILRGTQDGLLSLEDNEAQQLPPYENYYQNLGGDDNIHRSYELLACFKKDMHKVETYLTVAKCRRSL EANCTL Yellow grouper growthhormone ITDGQRLFSIAVSRVQHLHLLAQRLFSDFESSLQTE (SEQ ID NO:21)EQRQLNKIFLQDFCNSDYIISPIDKHETQRSSVLKLLSISYRLVESWEFPSRSLSGGSAPRNQIFPKLSELKTGILLLIRANQDGAELFPDSSALQLAPYGNYYQSLGADESLRRTYELLACFKKDMHKVETYLTVAKCRLSPE ANCTL

[0010] Accordingly, the invention features a substantially purepolypeptide having an amino acid sequence at least 75% (e.g., 80, 85,90, 95, or 100%) identical to SEQ ID NO:2, at least 97% (e.g., 98, 99,or 100%) identical to SEQ ID NO:6, or at least 92% (e.g., 95, 98, or100%) identical to SEQ ID NO:10. The polypeptide increases cellproliferation, i.e., when a cell is contacted with the polypeptide invivo or in vitro, the cell exhibits or will exhibit increasedproliferation in comparison to a cell without contact with thepolypeptide. Also featured is (1) a substantially pure polypeptideencoded by a nucleic acid that hybridizes under stringent conditions toa nucleic acid consisting of any one of SEQ ID NOs:4, 8, and 12; and (2)a substantially pure polypeptide having any one of SEQ ID NOs:2, 6, and10 with up to 10 (e.g., 2, 4, 5, 6, or 8) conservative amino acidsubstitutions.

[0011] The invention includes (1) an isolated nucleic acid whichhybridizes under stringent conditions to any one of SEQ ID NOs:4, 8, and12, e.g., a nucleic acid that encodes a polypeptide that increases cellproliferation; (2) an isolated nucleic acid which hybridizes understringent conditions to any one of SEQ ID NOs:1, 5, and 9, where thenucleic acid is at least 20 (e.g., at least 30, 40, 50, 100, 200, and300) nucleotides in length and does not contain 10 consecutive adenineresidues; and (3) a nucleic acid encoding any polypeptide of theinvention. A nucleic acid of the invention can also be less than 10,000,1000, 500, 100, 50, or 25 thousand nucleotides in length.

[0012] In addition, the invention features a transgenic fish whosegenomic DNA comprises a foreign sequence encoding a polypeptide of theinvention, wherein the transgenic fish exhibits increased cellproliferation as compared to a reference fish whose genomic DNA does nothave the foreign sequence. The transgenic fish can be a salmoniform(e.g., a member of the genus Plecoglossus), a perciform (e.g., a memberof the genus Epinephelus), or a cypriniform (e.g., a member of the genusDanio).

[0013] The term “substantially pure” as used herein in reference to agiven polypeptide means that the polypeptide is substantially free fromother biological macromolecules. The substantially pure polypeptide isat least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight.Purity can be measured by any appropriate standard method, for example,by column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

[0014] A “conservative amino acid substitution” is one in which an aminoacid residue is replaced with another residue having a chemicallysimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

[0015] By hybridization under “stringent conditions” is meanthybridization at 65° C., 0.5×SSC, followed by washing at 45° C.,0.1×SSC.

[0016] The “percent identity” of two amino acid sequences or of twonucleic acids is determined using the algorithm of Karlin and Altschul(Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlinand Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotidesearches are performed with the NBLAST program, score=100,wordlength=12. BLAST protein searches are performed with the XBLASTprogram, score=50, wordlength=3. Where gaps exist between two sequences,Gapped BLAST is utilized as described in Altschul et al. (Nucleic AcidsRes. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.

[0017] An “isolated nucleic acid” is a nucleic acid the structure ofwhich is not identical to that of any naturally occurring nucleic acidor to that of any fragment of a naturally occurring genomic nucleic acidspanning more than three separate genes. The term therefore covers, forexample, (a) a DNA which has the sequence of part of a naturallyoccurring genomic DNA molecule but is not flanked by both of the codingsequences that flank that part of the molecule in the genome of theorganism in which it naturally occurs; (b) a nucleic acid incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. Specifically excluded from this definition are nucleicacids present in mixtures of different (i) DNA molecules, (ii)transfected cells, or (iii) cell clones: e.g., as these occur in a DNAlibrary such as a cDNA or genomic DNA library.

[0018] By “cell proliferation” is meant an increase in cell number orcell size.

[0019] By “foreign sequence” as applied to a transgenic fish is meant anucleotide sequence that does not naturally occur in the parent fishfrom which the transgenic fish was derived. Thus, a foreign sequenceincludes a non-naturally occurring additional copy of a sequence foundin the parent fish or a new sequence of a fish species different fromthe parent fish species.

[0020] Other features or advantages of the present invention will beapparent from the following detailed description, and also from theclaims.

DETAILED DESCRIPTION

[0021] The invention relates to new fish growth hormones and their usein promoting cell proliferation and isolating other related growthhormone genes. Contemplated within the scope of the invention aretransgenic fish harboring a gene that expresses a polypeptide of theinvention. These fish exhibit increased cell proliferation as comparedto a non-transgenic parent fish. The production of transgenic fish iswell known in the art and can be produced using electroporation (Inoueet al., Cell Diff. Dev. 29:123-128, 1990; Müller et al., Mol. MarineBiol. Biotech. 1:276-281, 1992; and Patil et al., J. Exp. Zoology274:121-129, 1996), particle bombardment (Zelenin et al., FEBS287:118-120, 1991), Baekonization (Zhao et al., Mol. Marine Biol.Biotech. 2:63-69, 1993), or the procedures in the Examples below.

[0022] As well known in the art, all growth hormone genes involved froma common ancestral gene and, thus, have identical common structuralsequences, i.e., four helix bundles. See, for example, Watahiki et al.(1989) J. Biol. Chem. 264: 5535-5539. For zebrafish growth hormone, thesequences of four helix bundles are: Helix oneNQRLFNNAVIRVQHLHQLAAKMINDFEEGLMPEERR (SEQ ID NO:22) QLSKI Helix twoSSMLKLLRISFRLIESWEF (SEQ ID NO:23) Helix three KLADLKMGISVLIKGC (SEQ IDNO:24) Helix four DTSLRESFRLLACFKKDMHKVETYLRVANCR (SEQ ID NO:25) For ayugrowth hormone, the sequences of four helix bundles are: Helix oneNQRLFSIAVNRAQHLHLLAQKMFNDFEGNLSPDDRR (SEQ ID NO:26) QMNKI Helix twoSSVQKLLHISFRLVESWEY (SEQ ID NO:27) Helix three LKLTDLKLGIDTILRGT (SEQ IDNO:28) Helix four DDNIHRSYELLACFKKDMHKVETYLTVAKCR (SEQ ID NO:29) Foryellow grouper growth hormone, the sequences of four helix bundles are:Helix one GQRLFSIAVSRVQHLHLLAQRLFSDFESSLQTEEQR (SEQ ID NO:30) QLNKIHelix two SSVLKLLSISYRLVESWEF (SEQ ID NO:31) Helix threeKLSELKTGILLLIRAN (SEQ ID NO:32) Helix fourDESLRRTYELLACFKKDMHKVETYLTVAKCR (SEQ ID NO:33)

[0023] The following scheme shows sequences (boxed) that are essentialfor receptor binding, and thus, cell proliferation stimulation. Notethat asterisks indicate identical amino acids in all three sequences.               10        20        30        40        50        60        70          ----|----|----|----|----|----|----|----|----|----|----|----|----|----|----|DRGH MARAKVKKQKVVVSKKVBQGJASDBQRK{overscore(|FNNAVIRVQHLH|)}QLAAKMINDFEEGLMPE                                 {overscore(|            |)}                  PaGHMGQVLLLVTLLLVSDLVRSASGSENQRL{overscore(|FSIAVNRAQHLH|)}LLAQKMFNDFEGNLSPD                                 {overscore(|            |)}                  EaGHMDRVVLLLS--VVSLGVSSQPITDGQRL{overscore(|FSIAVSRVQHLH|)}LLQRLFSDFESSLQTEE                                 {overscore(|            |)}                      *     *     **  *        ****  ** * **** **     ***  *     **  ***   ***** *    80        90       100       110----|----|----|----|----|----|----|----| ER{overscore(|RQLSK|)}IFPLSFC{overscore (|NSDSIETPTGKDETQ|)}KSSMLKLLRISFRL  {overscore (|     |)}       {overscore(|               |)}               DR{overscore(|RQMNK|)}IFLQDFC{overscore (|NSDSIIDPVDKHETQ|)}KSSVQKLLHISFRL  {overscore (|     |)}       {overscore(|               |)}               QL{overscore(|RQLNK|)}IFLQDFC{overscore (|NSDYIISPIDKHETQ|)}RSSVLKLLSISYRL  {overscore (|     |)}       {overscore(|               |)}                 *  * ***  **  *** ** ** **** **  **IESWEFPSQTLSSTI                 VESWEYPSQALSSSL                VESWEFPSRSLSGG-                        120       130       140       150       160       170       180       190            ----|----|----|----|----|----|----|----|----|----|----|----|----|----|----|----|---DRGH SNSLTI{overscore(|GNPNQLTEKLADLKMGISV|)}LIKGCLDGQPNMDDNDSLPL-PFEOFYLTVG-DTSLRES (SEQ IDNO 10)            {overscore(|                   |)}                                        PaGHSLSRFS{overscore(|EIPL----KLTDLKLGIDT|)}ILRGTQDGLLSLEDNEAQQLPPYENYYQNLGGDDNIHRS (SEQ IDNO 2)            {overscore(|                   |)}                                        EaGHSAPRNQ{overscore(|IFP-----KLSELKTGILL|)}LIRANQDGAELFPDSSALQLAPYGNYYQSLGADESLRRT (SEQ IDNO 6)            {overscore(|                   |)}                                            *       *     **  ** **        **     * *   *   *       **************** ** ** *  *200       210 -|----|----|-- FRLL{overscore(|ACFKKDMHKVETYLRV|)}ANCRRSLDSNCTL     {overscore(|                |)}              YELL{overscore(|ACFKKDMHKVETYLTV|)}AKCRRSLEANCTL     {overscore(|                |)}              YELL{overscore(|ACFKKDMHKVETYLTV|)}AKCRLSPEANCTL     {overscore(|                |)}             ***                                                 

[0024] Without further elaboration, it is believed that one skilled inthe art can, based on the above disclosure, the isolation of growthhormone genes described below, and the production of transgenic fishdescribed below, utilize the present invention to its fullest extent.The following examples are to be construed as merely illustrative of howone skilled in the art can isolate and use the polypeptides and nucleicacids of the invention, and are not limitative of the remainder of thedisclosure in any way. Any publications cited in this disclosure arehereby incorporated by reference.

EXAMPLE

[0025] The isolation and characterization of fish growth hormone geneswere carried out using the following methods.

[0026] Molecular cloning of growth hormone genes. About one hundredpituitary glands each of ayu and yellow grouper were collected. mRNA(about 5 mg) were isolated from pituitary glands using an mRNA isolationkit (Stratagene). cDNA were synthesis using a cDNA library synthesis kit(Stratagene). The inserts of interest were cloned into a ZAP expressionvector (Stratagene) and packaged to produce ayu and yellow grouperpituitary cDNA libraries.

[0027] A pair of degenerated primers were designed based on thesequences of common carp, tuna, red sea bream, rainbow trout, yellowtail, and chum salmon growth hormone genes. mRNA was extracted frompituitary glands of ayu, yellow grouper, and zebrafish, and thecorresponding growth hormone cDNA were amplified from the mRNA usingRT-PCR and the degenerate primers. Labelled probes were then producedfrom these amplified fragments using a random primer synthesis kit(RediPrime II, Amersham). The probes were used to screen the ayu andyellow grouper pituitary cDNA libraries, as well as a zebrafish headcDNA library. Purified positive clones were sequenced using an ABIauto-sequencer model 377.

[0028] Expression of new growth hormones. For the sake of simplicity,the ayu, yellow grouper, and zebrafish recombinant growth hormones willbe abbreviated rPaGH, rEaGH, and rDrGH, respectively. Growth hormonecDNA clones were PCR amplified using primers designed to containconvenient restriction sites. The amplified fragments were digested withNdeI and XhoI and cloned into similarly digested PET-20b(+) (Novagen).The resulting vectors were then used to transform E. coliBL21(DE3)pLysS.

[0029] Transformed host cells growing to an OD₆₀₀ of 0.3 were inducedwith 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG) and thenincubated at 37° C. for 16 hours. The induced proteins were isolated andexamined using SDS-PAGE. The SDS-PAGE bands were then excised from thegel and sequenced using an ABI model 477 protein sequencer to ensurethat the recombinantly produced proteins were authentic.

[0030] Recombinant protein isolation. Inclusion bodies were isolatedfrom transformed bacteria generally using the methods described in Gizaet al., Gene 78:73-84, 1989. Induced bacteria were harvested bycentrifugation at 4000×g for 30 minutes. The cell pellet was thensuspended in and mixed well with ice cold lysis buffer 1 (50 mM Tris [pH7.8], 200 mM NaCl, 0.1 mM EDTA, 5% glycerol). To the mixture was thenadded {fraction (1/10)} volume cell lysis buffer 2 (50 mM EDTA, 10%Triton X-100). Finally, lysozyme was added to a final concentration 10μg/ml to facilitate cell wall breakage. The reaction mixture was put onice for 3 hours. To facilitate cell wall breakage, the mixture wassonicated immediately after the incubation. DNA was digested with DNaseI at 37° C. for 1 hour. Steps were repeated as necessary to ensurecomplete lysis of the host cells.

[0031] Total cell lysate was separated into soluble and insolubleportions by centrifugation at 4000×g for 30 minutes. The insolublepellet (protein) was collected and washed twice with ice cold, doubledistilled water. The precipitated protein was denatured and renatured asdescribed in Giza et al., Gene 78:73-84, 1989. The pellet was dissolvedin 30 ml of 4.5 M urea, 10 mM Tris-HCl, and 0.1 M β-mercaptoethanol. ThepH of the mixture was raised to about 11-13 by adding NaOH as needed, todissolve the pellet.

[0032] Quantitation and secondary structure determination of recombinantgrowth hormones. For quantitation of recombinantly produced fish growthhormone, an extinction coefficient of 700 g⁻¹ cm⁻¹ at 277 nm wasassumed. This extinction coefficient is characteristic of bovine growthhormone (Burger et al., J. Biol. Chem. 241:449-457, 1966). Theconcentration of rPaGH and rEaGH was estimated by UV absorbance at 277nm.

[0033] Circular dichroism (CD) spectra were determined on a Jasco J-720Cspectropolarimeter. All spectra were determined at room temperatureusing a cuvette with 0.1 cm path length. Data were expressed as the meanresidue mole ellipticity (θ) (deg·cm²/dmole), and were based on amolecular weight of 22 KD.

[0034] Cell proliferation assay. The ability of recombinant proteins tostimulate cell proliferation was examined using a MTS-formazanconversion assay (Promega). 10⁴ ZFL cells (Zebrafish liver cell line,described in Ghosh et al., Cell. Biol. Toxicol. 10:167-176, 1994) in logstage growth were seeded on 96-well plates. Various amounts ofrecombinant or control proteins were added into 100 μl culture medium(L15 [Gibco BRL] supplemented with 0.5% fetal bovine serum and 0.1%bovine serum albumin) to achieve a final concentration of recombinantgrowth hormone or control proteins ranging from 0.1 to 1'10⁶ pM. Thecells were then placed in contact with the recombinant proteins at 28°C. for 48 hours.

[0035] After the 48 hours incubation, 20 μl of CellTiter 96 Aqueous onesolution (Promega) was added into each well, and the plates wereincubated for 4 hours at 37° C. in a humidified, 5% CO₂ incubator. Theabsorbance of each well at 450 nm was recorded using a plate reader.CHO-K1 (Chinese hamster ovary) cells, which do not express a growthhormone receptor, was used as a control for effects independent fromphysiological binding of a growth hormone to its receptor on a cell.

[0036] Transgenic fish. Fish growth hormone cDNA was inserted into a CMVexpression vector as follows. The fish pituitary cDNA was synthesizedusing a ZAP-cDNA® synthesis kit (Stratagene, cat. no. 200400) andinserted into library vectors using the ZAP Express® vector XR kit(Stratagene, cat. no. 239213). The expression vector was then introducedinto fish sperm cells using a Baekon 2000 electroporator. The settingsfor electroporation were 10 kV, 64 pulses, 4 cycles, 0.4 μs burst time,and 160 μs pulse time. Ten microliters of harvested fish seminal fluidcontaining sperm were mixed with 0.5 ml saline containing 100 μg/ml fishgrowth hormone cDNA expression vector. After electroporation, the spermwas mixed with mature fish eggs and saline for fertilization. Thezygotes were incubated in 16 to 18° C. fresh water until hatching.

[0037] The gene transduction efficiency was evaluated by extractinggenomic DNA from hatched fish and amplifying the transgene using primershybridizing to the CMV promoter sequence and the introduced growthhormone sequence. The transgene was confirmed by the size andrestriction enzyme digestion pattern of the amplified PCR fragment.

[0038] The results of this Example are described below.

Degenerate Primers for Cloning Fish Growth Hormones

[0039] Using the growth hormone gene sequences of the common carp, tuna,red sea bream, rainbow trout, yellow tail, and chum salmon, a consensusgrowth hormone sequence was generated and used to design two primers.The sense primer was CAAMAYCTBCACCWRYTSGCYSMR (SEQ ID NO:13) andantisense primer was CTTGTGCATGTCYTTYTTRAARCA (SEQ ID NO:14). The singleletter codes for degenerate nucleotides are M=A or C; R=A or G; W=A orT; S=C or G; Y=C or T; K=G or T; V=A, C, or G; H=A, C, or T; D=A, G, orT; and B=C, G, or T. These primers were used to amplify a 456 bp, 450bp, and 462 bp PCR product from ayu, yellow grouper, and zebrafish cDNA,respectively. These PCR fragments were used to synthesis DNA probes forcloning the complete full length growth hormone cDNA of ayu, yellowgrouper, and zebrafish.

Molecular Cloning of Growth Hormone Gene From Ayu Pituitary Gland cDNALibrary

[0040] A full length DNA clone of ayu growth hormone was isolated byscreening an ayu pituitary cDNA library with ayu growth hormone DNAprobes produced in Example 1. The nucleotide sequences of the ayu growthhormone cDNA clone was 1410 bp in length and encoded 208 amino acids(see SEQ ID NOs:1 and 2 above). The 5′ untranslated region was 68 bp inlength, and the 3′ untranslated region was 718 bp in length. A 22 aminoacids signal peptide was predicted using the software program SignalP(Nielsen et al., Protein Eng. 10:1-6, 1997).

Molecular Cloning of Growth Hormone Gene From a Yellow Grouper PituitaryGland cDNA Library

[0041] A full length DNA clone of yellow grouper growth hormone wasisolated by screening a yellow grouper pituitary cDNA library with theyellow grouper growth hormone DNA probes produced in Example 1. Thenucleotide sequence of yellow grouper growth hormone cDNA clone was 972bp in length, encoding 204 amino acid (see SEQ ID NOs:5 and 6). The 5′untranslated region was 69 bp in length and the 3′ untranslated regionwas 291 bp in length. A 19 amino acid signal peptide was predicted usingthe software program SignalP (Nielsen et al., supra).

Molecular Cloning of Zebrafish Growth Hormone Gene

[0042] A full length DNA clone of zebrafish growth hormone was isolatedby screening a zebrafish head cDNA library using the zebrafish growthhormone probes produced in Example 1. The nucleotide sequence of thezebrafish growth hormone cDNA clone was 1195 bp in length, encoding 210amino acids (see SEQ ID NOs:9 and 10). The 5′ untranslated region was 37bp in length, and the 3′ untranslated region was 528 bp in length. A 22amino acids signal peptide was predicted by the software program SignalP(Nielsen et al., supra)

Expression of Recombinant Ayu Growth Hormone

[0043] The DNA fragment encoding the mature ayu growth hormone wascloned into the PET-20b(+) expression vector (Novagen) to produce anopen reading frame encoding the ayu growth hormone with an extramethionine at the amino terminus and extra Leu-Glu-His₆ (SEQ ID NO:15)at the carboxyl terminus. The DNA fragment inserted into the expressionvector was PCR-amplified using primers AATTCCATATGTCAGAGAACCAGCGTGTA(SEQ ID NO:16) and CCGCTCGAGCAGGGTACAGTTGGCTTC (SEQ ID NO:17).

[0044] The rPaGH was produce in transformed bacteria induced with IPTGCell lysates were divided into soluble and insoluble portions. Analysisof protein fractions by SDS-PAGE indicated that most of the recombinantprotein was in the insoluble fraction. The molecular weight of rPaGH onSDS-PAGE was about 28 KD. The purity of rPaGH was about 74% of totalinsoluble protein, as determined by optical density. rPaGH wasdissolvable in a reducing solvent containing 0.1 M β-mercaptoethanol,4.5 M urea, and 5% glycerol (pH 12.4). The rPaGH was renatured byfour-step equilibrium dialysis. The four steps were (1) dialysis for 48hours in 10 mM Tris-HCl (pH 11), 1 M urea, 0.1 mM β-mercaptoethanol, 1mM cysteine, and 5% glycerol; (2) dialysis for 24 hours in 10 mMTris-HCl (pH 11), 0.1 mM β-mercaptoethanol, 1 mM cysteine, and 5%glycerol; (3) dialysis for 12 hours in 10 mM Tris-HCl (pH 8.8), 5%glycerol, and 0.1 M β-mercaptoethanol; and (4) dialysis for 12 hours in10 mM Tris-HCl (pH 8.8) and 0.1 M β-mercaptoethanol.

Expression of Recombinant Yellow Grouper Growth Hormone

[0045] The DNA fragment encoding the mature yellow grouper growthhormone was cloned into the PET-20b(+) expression vector (Novagen) toproduce an open reading frame encoding the yellow grouper growth hormonewith an extra methionine at the amino terminus and extra Leu-Glu-His₆(SEQ ID NO:15) at the carboxyl terminus. The DNA fragment inserted intothe expression vector was PCR-amplified using primersAATTCCATATGATCACAGACGGCCAGCGACTG (SEQ ID NO:18) andCCGCTCGAGCAGGGTACGGTTGGCCTCAGG (SEQ ID NO:10).

[0046] The rEaGH was produce in transformed bacteria induced with IPTG.Cell lysates were divided into soluble and insoluble portions. Analysisof protein fractions by SDS-PAGE indicated that most of the recombinantprotein was in the insoluble fraction. The molecular weight of rEaGH onSDS-PAGE was about 24 KD. The purity of rEaGH was about 90% of totalinsoluble protein, as determined by optical density. rEaGH wasdissolvable in a reducing solvent containing 0.1 M β-mercaptoethanol,4.5 M urea, and 5% glycerol (pH 12.6). The rEaGH was renatured by thefour-step equilibrium dialysis procedure described in Example 5 above.

Characterization of Growth Hormone Folding

[0047] CD spectra studies indicated that there were two broad negativeabsorption peaks at 218 nm and 211 nm and one positive absorption peakat 190 nm for both the refolded ayu and yellow grouper recombinantgrowth hormones. This is consistent with what is observed for native,purified human growth hormone. Similar spectra were observed for rEaGH,except that there were three negative absorption peaks at 254 nm, 263nm, and 270 nm. Therefore, based on Yang's estimation (Yang et al.,Methods Enzymol. 13:208-269, 1986), the secondary structure of foldedrPaGH contained about 36% helix, 34% β-sheet, 5.1% β-turn, and 25.7%random coil, the RMS value being 8.36. It was also estimated that thesecondary structure of folded rEaGH contained about 59% helix, 0%β-sheet, 24.6% β-turn, and 16.5% random coil, the RMS value being 12.59.

Bioactivity of Recombinant Growth Hormones

[0048] The bioactivity of recombinant growth hormones was evaluatedusing ZFL cells and a MTS-formazan conversion assay. A concentration of1 μm, human growth hormone, rPaGH, and rEaGH increased cellproliferation, relative to no growth hormone added, by 13.7%, 118.3%,and 58.3%, respectively. In addition, the increase in cell proliferationwas dose dependent in the range from about 0.1 to 10⁶ pM. Theproliferation of a CHO control cell line was not affected by theaddition of any growth hormone, as expected, because these cells do notexpress a growth hormone receptor.

Transgenic Ayu Overexpressing Ayu Growth Hormone

[0049] Ayu growth hormone cDNA was inserted into a CMV expression vectoras described above. The expression vector was then introduced into ayusperm cells, which were used to fertilize ayu fish eggs. After about 10to 12 days, the fertilized eggs produced hatchlings. One hundred hatchedayu were sacrificed, and their genomic DNA was extracted. Eighty six ofthe hundred contained the CMV-ayu growth hormone expression cassette intheir genomic DNA. The presence of the expression cassette was confirmedby the size of a DNA fragment amplified from the genomic DNA and by theBamHI I digestion pattern.

Other Embodiments

[0050] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope ofthis invention.

What is claimed is:
 1. A substantially pure polypeptide comprising anamino acid sequence at least 75% identical to SEQ ID NO:2, at least 97%identical to SEQ ID NO:6, or at least 95% identical to SEQ ID NO:10,wherein the polypeptide increases cell proliferation.
 2. The polypeptideof claim 1, wherein the amino acid sequence is selected from the groupconsisting of SEQ ID NOs:2, 6, and
 10. 3. A substantially purepolypeptide encoded by a nucleic acid that hybridizes under stringentconditions to a single stranded nucleic acid consisting of any one ofSEQ ID NOs:4, 8, and 12, wherein the polypeptide increases cellproliferation.
 4. A substantially pure polypeptide comprising any one ofSEQ ID NOs:2, 6, and 10 with up to 10 conservative amino acidsubstitutions, wherein the polypeptide increases cell proliferation. 5.The polypeptide of claim 4, wherein the polypeptide comprises any one ofSEQ ID NOs:2, 6, and 10 with up to 5 conservative amino acidsubstitutions.